Cytotoxic T Lymphocyte Inducing Immunogens For Prevention Treatment and Diagnosis of Dengue Virus Infection

ABSTRACT

Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHR) are significant global public health problems and understanding the overall immune response to infection will contribute to appropriate management of the disease and its potentially severe complications. Live attenuated and subunit vaccine candidates, which are under clinical evaluation, induce primarily an antibody response to the virus and minimal cross-reactive T cell responses. Currently, there are no available tools to assess protective T cell responses during infection or post vaccination. The present invention incorporates immunoproteomics to uncover novel HLA-A2 specific epitopes derived from Dengue Virus (DV)-infected cells. These epitopes are conserved with epitope-specific CTLs cross-reacting against all four DV serotypes. These epitopes have potential as new informational and diagnostic tools to characterize T cell immunity in Dengue virus (DV) infection, and serves as a universal vaccine candidate complementary to current vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of US national application 14/116,filed on 29 Dec. 2014 now allowed, which is the US national phaseapplication of PCT/US2012/044625, filed on 28 Jun. 2012 and which claimspriority to U.S. Provisional Application No. 61/502,365, filed on 29Jun. 2011, now expired, the disclosure of which is herein incorporatedby reference in its entirety.

This invention was made with Government support under Grant NumberA1062177 awarded by the National Institutes of Health, The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunogens whosestructures incorporate polypeptides comprising epitopic peptides derivedfrom proteins expressed by dengue virus (DV 1-4) infected cells and usesof said immunogens in eliciting cytotoxic T lymphocyte (CTL) responsesfor the diagnosis, prevention and treatment of all 4 serotypes of denguevirus infection.

BACKGROUND OF THE INVENTION

Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) are significantpublic health problems internationally, and caused by four antigenicallydistinct serotypes of dengue virus (DV1-4). Approximately 36 millioncases of DF and 2.1 million cases of DHF occur annually and 2.5-3.5billion of the world population are at risk of transmission of DF.Although patients who have recovered from DV infection are immune tore-challenge with the same serotype, secondary infection with adifferent DV serotype can lead to increased risk of DHF and dengue shocksyndrome (DSS). The DV genome consists of structural and non-structuralproteins with DV serotypes 1-4 having approximately 60% 74% sequencehomology in the E gene, which can induce cross-reacting antibodies.

Considerable effort has been devoted to the development of effectivevaccines against DV. Live attenuated viruses, inactivated viruses,recombinant proteins, chimeric viruses, DNA vaccines, and syntheticpeptides are being evaluated in the clinic. Due to the lack of an animalmodel or in vitro markers for attenuation in humans, chimeric vaccineswith two or less dengue antigens, which results in limited T cellimmunity, are being pursued. While antibodies against one serotype canbe neutralizing and protective, risk of DHF after exposure to differentserotypes has been observed. Early vaccine studies also demonstrated Tcell responses to DV, but they were largely DV serotype specific. Thismay suggest that the level of presentation of MHC class I (MHCI) andclass II (MHCII) antigens differs among serotypes. Beneficial effects ofthe vaccine-induced Th1 response further underscore the significance ofthe T cell response in vaccine development. The weight of evidencesuggests that a useful Dengue virus vaccine will require both B- andT-cell responses to not only successfully protect against infection byeach of the four serotypes, but also against the complications ofantibody dependent enhancement (ADE).

The present disclosure involves peptides that are associated with theHLA-A2, HLA-A24, or HLA-B7 molecules, HLA-A2 supertypes, HLA-A24supertypes, and HLA-B7 supertypes. A supertype is a group of HLAmolecules that present at least one shared epitope. The presentdisclosure involves peptides that are associated with HLA molecules, andwith the genes and proteins from which these peptides are derived.

Three different methodologies have typically been used for identifyingthe peptides that are recognized by CTLs in infectious disease field.These are: (1) the genetic method; (2) motif analysis: (3) theimmunological and analytical chemistry methods or the Immunoproteomicsmethod.

The genetic method is an approach in which progressively smaller subsetsof cDNA libraries from diseased cells are transfected into cells thatexpress the appropriate MHC molecule but not the disease-specificepitope. The molecular clones encoding T cell epitopes are identified bytheir ability to reconstitute disease specific T cell recognition oftransfected cells. The exact T cell epitope is then identified by acombination of molecular subcloning and the use of synthetic peptidesbased on the predicted amino acid sequence. Such methods, however, aresusceptible to inadvertent identification of cross-reacting peptides,and are not capable of identifying important post-translationalmodifications.

Motif analysis involves scanning a protein for peptides containing knownclass I MHC binding motifs, followed by synthesis and assay of thepredicted peptides for their ability to be recognized bydisease-specific CTL. This approach requires prior knowledge of theprotein from which the peptides are derived and widely used in virus orbacterial infection field. This approach is also greatly hampered by thefact that not all of the predicted peptide epitopes are presented on thesurface of a cell (Yewdell, J. W. and Bennink, J. R., Ann.Rev.Immunol.,17:51-88, (1999)), thus additional extensive experimentation is requiredto determine which of the predicted epitopes is useful.

Immunoproteomics method involves a combination of cellular immunologyand mass spectrometry. This approach involves the actual identificationof endogenous CTL epitopes present on the cell surface by sequencing thenaturally occurring peptides associated with class I MHC molecules. Inthis approach, cells are first lysed in a detergent solution, thepeptides associated with the class I MHC molecules are purified, and thepeptides are fractionated by high performance liquid chromatography(HPLC). Peptide sequencing is readily performed by tandem massspectrometry (Henderson, R. A. et al., Proc.Natl.Acad.Sci.U.S.A,90:10275-10279, (1993).

A number of recent reports for different types of virus infectionsprovide evidence that CTL specific for epitopes that are naturallyprocessed and presented by infected cells have markedly greater impacton the control of virus replication. Undoubtedly, CTLs have been shownto play an important role in the elimination of dengue virus-infectedcells. Thus, identification of antigenic peptides that are presented byinfected cells and recognized by epitope-specific CTLs may suggest newways to suppress viral replication and prevent persistent infection.Multiple peptides from conserved regions of dengue virus may proveessential in the development of a universally immunogenic vaccine. Inrecent years, several MHC class I specific peptides have been reportedby the screening of algorithm-predicted T-cell epitopes using T cellsfrom individuals participating in experimental DV vaccine trials as wellas those infected with DV. However, these peptides were not subsequentlyinvestigated nor determined to be presented by DV infected cells.

Little is known about cross serotype conserved T cell epitopes that areimmunologically relevant in eliciting an effective T cell response tothe four DV serotypes. Several groups have attempted to identify T cellepitopes by either motif prediction of MHC binding peptides from Dengueproteins, or by screening overlapping peptides from structural andnonstructural Dengue proteins. Screening PBMCs from individuals in a DVvaccine trial and DV-infected patients using a panel ofalgorithm-derived peptide sequences identified a few DV serotypespecific T cell epitopes. However, a comprehensive analysis of naturallypresented epitopes on infected cells has never been undertaken orreported.

SUMMARY OF THE INVENTION

The present invention relates to immunogens comprising polypeptides withamino acid sequences comprising epitopic sequences selected from thesequences of SEQ ID NO: 1-17 and which immunogens facilitate a cytotoxicT lymphocyte (CTL)-mediated immune response against various serotypespecific dengue virus (DV) infection. The present invention also relatesto nucleic acid molecules that encode for the polypeptides theirisoforms and splice variants from which the polypeptides are derived, ofsuch immunogens, and which can also be used to facilitate an immuneresponse against DV.

The present invention provides compositions comprising the immunogendescribed herein, and polynucleotides that direct the synthesis of suchpolypeptides, whereby the oligopeptides and polypeptides of suchimmunogens are capable of inducing a CTL response against cellsexpressing a protein comprising an epitopic sequence of at least one ofSEQ ID NO: 1-17. The cells are usually DV infected cells, preferablydengue virus serotypes 1-4 expressing such proteins.

The present invention further relates to polynucleotides comprising thegene coding for a polypeptide of the immunogens disclosed herein. Thepresent invention also provides methods that comprise contacting alymphocyte, especially a CTL, with an immunogen or its isoforms orsplice variants of the invention under conditions that induce a CTLresponse against a DV infected cell. The methods may involve contactingthe CTL with the immunogenic peptide in vivo, in which case thepeptides, polypeptides, and polynucleotides of the invention are used asvaccines, and will be delivered as a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or delivery system andthe immunogen, typically along with an adjuvant or one or morecytokines.

Alternatively, the immunogens of the present invention can be used toinduce a CTL response in vitro. The generated CTL can then be introducedinto a patient with DV infection, more specifically DV serotypes 1-4.Alternatively, the ability to generate CTL in vitro could serve as adiagnostic for DV infection generally, including dengue virus serotypes1-4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Dengue virus infection analysis. (A) JY, HepG2 cells as well asDC from healthy HLA-A2+ donors were infected with DV2 and DV3 virus andincubated for 72 hrs. Cells were lysed and immunoblotted for E protein.(B) HepG2 cells infected with DV2 virus. After 72 hr incubation, cellswere stained for E protein. Densitometry in arbitrary units (AU) usingOdyssey Infrared Imaging System software.

FIG. 2: Validation of naturally processed MHC class I peptides isolatedfrom dengue virus infected DC/JY cells. MS/MS spectra of identified MHCpeptides (Seq ID: 1-[P4], 2-[P2], 3-[P3], 15-[P1]) [P1A-P4A] and theirsynthetic analogs [P1B-P4B].

FIG. 3: CTL generated with DV epitopes are specific and cross-reactive.(A) T2 cells were pulsed with peptide and used as targets in an ELISpotassay with CTL that were generated from HLA-A2+ PBMCs against specificpeptides. (B). JY and HepG2 cells were infected with either DV2 or DV3and used as targets in an ELISpot assay. K562 were also used as NK celltarget cells. Results were normalized against peptide unpulsed controls.

FIG. 4: CTL generated with DV epitopes cross-react with Thai isolates ofDV. (A) HepG2 cells were infected with Thai isolates of DV and stainedfor E protein after 72 hr infection. Densitometry is in arbitrary units(AU), using Odyssey Infrared Imaging System software. (B) JY cells wereinfected with Thai isolates of DV. Following a 72 hr infection, cellswere lysed and immunoblotted for E protein. Either HepG2 (C) or JY (D)were infected with DV Thai isolates and used as targets in an ELISpotassay. CTL were generated against P1-4 derived from DV-infected cells.Results were normalized against negative (uninfected) controls.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein and except as noted otherwise, all terms are defined asgiven below. The term “peptide” is used herein to designate a series ofamino acid residues, connected one to the other typically by peptidebonds between the alpha-amino and carbonyl groups of the adjacent aminoacids. The peptides are typically 9 amino acids in length, but can be asshort as 8 amino acids in length, and as long as 14 amino acids inlength. The series of amino acids are consider an “oligopeptide” whenthe amino acid length is greater than about 14 amino acids in length,typically up to about 30 to 40 residues in length. When the amino acidresidue length exceeds 40 amino acid residues, the series of amino acidresidues is termed “polypeptide”.

A peptide, oligopeptide, polypeptide, protein, or polynucleotide codingfor such a molecule is “immunogenic” and thus an immunogen within thepresent invention if it is capable of inducing an immune response. Inthe present invention, immunogenicity is more specifically defined asthe ability to induce a CTL-mediated response. Thus, an immunogen wouldbe a molecule that is capable of inducing an immune response, and in thepresent invention, a molecule capable of inducing a CTL response. Animmunogen may have one or more isoforms or splice variants that haveequivalent biological and immunological activity, and are thus alsoconsidered for the purposes of this invention to be immunogenicequivalents of the original, natural polypeptide.

A T cell “epitope” is a short peptide molecule that binds to a class Ior II MHC molecule and that is subsequently recognized by a T cell. Tcell epitopes that bind to class I MHC molecules are typically 8-14amino acids in length, and most typically 9 amino acids in length.

Three different genetic loci encode for class I MHC molecules: HLA-A,HLA-B, and HLA-C. The present invention involves peptides that areassociated with HLA-A2 supertypes. A supertype is a group of HLAmolecules that present at least one shared epitope. MHC moleculepeptides that have been found to bind to one member of the MHC allelesupertype family (A2 for example) are thought to be likely to bind toother members of the same supertype family (A68 for example).

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides. The nucleotide sequence encoding for a particularpeptide, oligopeptide, or polypeptide naturally occurring orsynthetically constructed.

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region whoseexpression product retains essentially the same biological orimmunological function or activity as the expression product of thecomplete coding region.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring).

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a human, such immune response taking the form of stimulating aCTL response within the recipient. Alternatively, the “active fragment”may also be used to induce a CTL response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment.” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, the oligopeptidesresulting from such treatment would represent portions, segments orfragments of the starting polypeptide.

The term “percent identity” when referring to a sequence, means that asequence is compared to a described sequence after alignment of thesequence to be compared with the described sequence. The PercentIdentity is determined according to the following formula:

Percent Identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequence(“R”) and the Compared Sequence (“C”) over the length of alignmentbetween R and C wherein (i) each base or amino acid in R that does nothave a corresponding aligned base or amino acid in the C and (ii) eachgap in R and (iii) each aligned base or amino acid in R that isdifferent from an aligned base or amino acid in C, constitutes adifference; and R is the number of bases or amino acids over the lengthof the alignment with C with any gap created in R also being counted asa base or amino acid.

DESCRIPTION

The present invention embodies generally immunogens and immunogeniccompositions, and methods of use thereof, for the prevention, treatment,and diagnosis of dengue virus infection, including DV serotypes 1-4. Theimmunogens comprise proteins or polypeptides whose amino acid sequencesincludes one or more epitopic oligopeptides with sequences selected fromthe group SEQ ID NO: 1-17. In addition, the invention further embodiespolynucleotides that can be used to stimulate a CTL response against DVinfection, and more specifically serotypes DV 1-4.

One embodiment of the present invention includes compositions for DVpeptides, subsequence and portions thereof, nucleic acid sequencesencoding DV peptides, subsequences and portions thereof, and host cellsexpressing DV peptides, subsequences and portions thereof. Oneparticular aspect of the subsequence or portion of the DV polypeptidesequence includes epitopic peptides. These embodiments furtherincorporate useful pharmaceutical compositions such as, but not limitedto, an adjuvant (e.g., Freund's complete or incomplete adjuvant) oradministration with traditional prophylactic viral vaccine formulations(e.g., live attenuated viruses, inactivated viruses, recombinantproteins, chimeric viruses, DNA vaccines, and synthetic peptides).

The invention includes kits that contain DV peptides, subsequences andportions thereof, compositions, that optionally include instructions fortreating (prophylactic or therapeutic), vaccinating or immunizing asubject against a DV infection, or treating (prophylactic ortherapeutic) a subject having or at risk of having a Dengue virusinfection or pathology.

In accordance with further embodiments of the invention, methods fortreating a subject having a DV infection (acute) are provided. In oneembodiment, a method includes administering to a subject in need thereofan amount of a DV peptide or epitopic peptide, subsequence or portionthereof, sufficient to treat the subject for the pathogen infection.

In accordance with further embodiments of the invention, there areprovided prophylactic methods including methods of vaccinating andimmunizing a subject against a DV infection (acute) such as, but notlimited to, protecting a subject against a DV infection to decrease orreduce the probability of a DV infection or pathology in a subject or todecrease or reduce susceptibility of a subject to a DV infection orpathology or to inhibit or prevent a DV infection in a subject.

In accordance with further embodiments of the present invention specificoligopeptide sequences are disclosed with amino acid sequences shown inSEQ ID NO: 1-17 representing epitopic peptides (i.e. immunogenicoligopeptide sequences) of at least about 8 amino acids in length,preferably about 9 amino acids in length (i.e., nonapeptides), and nolonger than about 14 amino acids in length and present as part of alarger structure, such as a polypeptide or full length protein.

The polypeptides forming the immunogens of the present invention haveamino acid sequences that comprise at least one stretch, possibly two,or more stretches of about 8 to 10 or up to 14 residues in length andwhich stretches differ in amino acid sequence from the sequences of SEQID NO: 1-17 by no more than about 1 amino acid residue, preferably aconservative amino acid residue, especially amino acids of the samegeneral chemical character, such as where they are hydrophobic aminoacids.

These polypeptides are of any desired length so long as they haveimmunogenic activity in that they are able, under a given set ofdesirable conditions, to elicit in vitro or in vivo the activation ofcytotoxic T lymphocytes (CTLs) (i.e., a CTL response) against apresentation of DV specific protein, especially DV 1-4 specific proteinwhere said proteins are presented in vitro or in vivo by an antigenpresenting cell (APC). The proteins and polypeptides forming theimmunogens of the present invention can be naturally occurring orsynthesized chemically.

The present invention further embodies an isolated polypeptide,especially one having immunogenic activity, the sequence of whichcomprises within it one or more stretches comprising any 2 or more ofthe sequences of SEQ ID NO: 1-17 and in any relative quantities andwherein said sequences may differ by one amino acid residues from thesequences of SEQ ID NO: 1-17 in any given stretch of 8 to 10, or up to14 amino acid residues. Thus within the present invention, by way of anon-limiting example only, such polypeptide may contain as part of itsamino acid sequence, nonapeptide fragments having up to 8 amino acidsidentical to a sequence of SEQ ID NO: 1,2,7,8 such that the polypeptidecomprises, in a specific embodiment, 2 segments with at least 8 residuesidentical to SEQ ID NO: 1 and SEQ ID NO: 2 and one segment with at least8 residues identical to SEQ ID NO: 7. In other embodiments, othercombinations and permutations of the epitopic sequences disclosed hereinmay be part of an immunogen of the present invention or of such apolypeptide so long as any such polypeptide comprises at least 2 suchepitopes, whether such epitopes are different or the same.

All of the epitopic peptides of SEQ ID NO: 1 through 17 are derived fromproteins expressed by DV infected cells and sequences and wereidentified through the method of Immunoproteomics and Automated HighThrough-put Sequencing (HTPS).

In addition to the sequences of SEQ ID NO: 1-17, the proteins andpolypeptides forming the immunogens of the present invention furthercomprise one or more other immunogenic amino acid stretches known to beassociated with DV infection, and more specifically DV serotypes 1-4,and which may stimulate a CTL response whereby the immunogenic peptidesassociate with HLA-A2 or HLA-A24 or HLA-B7, HLA supertypes, or any class1 MHC (i.e., MHC-1) molecule.

The immunogens of the present invention can be in the form of acomposition of one or more of the different immunogens and wherein eachimmunogen is present in any desired relative abundance.

The oligopeptides and polypeptides useful in practicing the presentinvention may be derived by fractionation of naturally occurringproteins by methods such as protease treatment, or they may be producedby recombinant or synthetic methodologies that are well known and clearto the skilled artisan. The polypeptide may comprise a recombinant orsynthetic polypeptide having at least one of SEQ ID NO: 1-17. Thus,oligopeptides and polypeptides of the present invention have at leastone immunogenic peptides within the amino acid sequence of saidoligopeptides and polypeptides, and said immunogenic peptides, orepitopes, which are the same or different, or may have any number ofsuch sequences wherein some of them are identical to each other in aminoacid sequence and said epitopic sequences occur in any order within saidimmunogenic polypeptide sequence. The location of such sequences withinthe sequence of a polypeptide forming an immunogen may affect relativeimmunogenic activity. In addition, immunogens of the present inventionmay comprise more than one protein comprising the amino acid sequencesdisclosed herein. Such polypeptides may be part of a single compositionor may themselves be covalently or non-covalently linked to each other.

The immunogenic peptides disclosed herein may also be linked directlyto, or through a spacer or linker to: an immunogenic carrier such asserum albumin, tetanus toxoid, keyhole limpet hemocyanin, dextran, or arecombinant virus particle; an immunogenic peptide known to stimulate aT helper cell type immune response; a cytokine such as interferon gammaor GMCSF; a targeting agent such as an antibody or receptor ligand; astabilizing agent such as a lipid; or a conjugate of a plurality ofepitopes to a branched lysine core structure, such as the so-called“multiple antigenic peptide” described in (Posneft, D. N. et al.,J.Biol.Chem., 263:1719-1725, (1988)); a compound such as polyethyleneglycol to increase the half-life of the peptide; or additional aminoacids such as a leader or secretory sequence, or a sequence employed forthe purification of the mature sequence. Spacers and linkers typicallycomprise relatively small, neutral molecules. In addition, such linkersneed not be composed of amino acids but any oligomeric structures willdo as well so long as they provide the correct spacing so as to optimizethe desired level of immunogenic activity of the immunogens of thepresent invention. The immunogen may therefore take any form that iscapable of eliciting a CTL response.

Immunogens, such as proteins, oligopeptides and polypeptides of theinvention, are structures that contain the peptides disclosed accordingto the present invention but such immunogenic peptides may notnecessarily be attached thereto by the conventional means of usingordinary peptide bounds. The immunogens of the present invention simplycontain such peptides as part of their makeup, but how such peptides areto be combined to form the final immunogen is through any means known inthe art.

The peptides that are naturally processed and bound to a class I MHCmolecule, and which are recognized by the DV-specific CTL, need not bethe optimal peptides for stimulating a CTL response. Thus, the abilityto modify a peptide such that it more readily induces a CTL response isconsidered. Generally, the peptides may be modified at amino acidresidues that are predicted to interact with the class I MHC molecule,in which case the goal is to create a peptide that has a higher affinityfor the class I MHC molecule than does the original peptide. Thepeptides can be modified at amino acid residues that are predicted tointeract with the T cell receptor on the CTL, in which case the goal isto create a peptide that has a higher affinity for the T cell receptorthan does the original peptide. Both of these types of modifications canresult in a variant peptide that is related to an original peptide, butwhich is better able to induce a CTL response than is the originalpeptide as selected from SEQ ID NO: 1-17.

The original peptides disclosed herein can be further modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain. Such substitutions can be conservative.Less conservative substitutions or even highly non-conservativereplacements are also considered since chemical effects are not totallypredictable.

Based on cytotoxicity assays, an epitope is considered substantiallyidentical to the reference peptide if it has at least 10% of theantigenic activity of the reference peptide as defined by the ability ofthe substituted peptide to reconstitute the epitope recognized by a CTLin comparison to the reference peptide. Thus, when comparing the lyticactivity in the linear portion of the effector:target curves withequimolar concentrations of the reference and substituted peptides, theobserved percent specific killing of the target cells incubated with thesubstituted peptide should be equal to that of the reference peptide atan effector:target ratio that is no greater than 10-fold above thereference peptide effector:target ratio at which the comparison is beingmade.

Preferably, when the CTLs specific for a peptide of SEQ ID NO: 1-17 aretested against the substituted peptides, the peptide concentration atwhich the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 μM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from at least two or more individuals, preferablythree.

Thus, the epitopes of the present invention may be identical tonaturally occurring DV infected cell-associated or DV-specific epitopesor may include epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

It should be appreciated that an immunogen may comprise one or morepeptides from SEQ ID NO: 1-17, a plurality of peptides selected from SEQID NO: 1-17, or comprise a polypeptide that itself comprises one or moreof the epitopic peptides of SEQ ID NO: 1-17.

The immunogenic peptides and polypeptides of the invention can beprepared synthetically, or any means known in the art, including thosetechniques involving recombinant DNA technology.

The coding sequences for peptides of the length contemplated herein canbe synthesized on commercially available automated DNA synthesizers ormodified to a desired amino acid substitution. The coding sequence canbe transformed or transfected into suitable hosts to produce the desiredfusion protein.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. Such cells can routinely be utilizedfor assaying CTL activity by having said genetically engineered, orrecombinant, host cells express the immunogenic peptides of the presentinvention.

The immunogenic peptides of the present invention may be used to elicitCTLs ex vivo from either healthy individuals or from DV infectedindividuals. Such responses are induced by incubating in tissue culturethe individual's CTL precursor lymphocytes together with a source ofantigen presenting cells and the appropriate immunogenic peptide. TheCTLs generated with peptides and polypeptides of the invention can beprepared by any means known in the art, including those techniquesinvolving ex vivo adoptive cell therapy technologies.

A variety of approaches are known in the art that allow polynucleotidesto be introduced and expressed in a cell, thus providing one or morepeptides of the invention to the class I MHC molecule binding pathway.Oligonucleotides that code for one or more of the peptides of theinvention can be provided to antigen presenting cells in such a fashionthat the peptides associate with class 1 MHC molecules and are presentedon the surface of the antigen presenting cell, and consequently areavailable to stimulate a CTL response.

By preparing the stimulator cells used to generate an in vitro CTLresponse in different ways, it is possible to control the peptidespecificity of CTL response. For example, the CTLs generated with aparticular peptide will necessarily be specific for that peptide.Likewise, CTLs that are generated with a polypeptide or polynucleotideexpressing or coding for particular peptides will be limited tospecificities that recognize those peptides. More broadly, stimulatorcells, and more specifically dendritic cells, can be incubated in thepresence of the whole parent protein. As a further alternative,stimulator cells, and more specifically dendritic cells, can betransduced or transfected with RNA or DNA comprising the polynucleotidesequence encoding the protein. Under these alternative conditions,peptide epitopes that are naturally cleaved out of the protein, andwhich are generated in addition to peptide epitopes of SEQ ID NO:1-17can associate with an appropriate class I MHC molecule, which may or maynot include HLA-A1, -A2, -A24, -B7. The selection of antigen presentingcells and the type of antigen with which to stimulate the CTL, is leftto the ordinary skilled artisan.

In certain embodiments, the methods of the present invention include amethod for inducing a CTL response in vitro that is specific for DVinfected cell expressing a molecule from A1, A2, A24, or B7 supertypes,whereby the method comprises contacting a CTL precursor lymphocyte withan antigen presenting cell that has bound an immunogen or hasexogenously acquired an immunogenic oligopeptide or polypeptidecomprising one or more of the peptides disclosed according to theinvention.

Another embodiment is directed to a process for inducing a CTL responsein vitro that is specific for DV infected cell expressing a moleculefrom A1, A2, A24, or B7 supertypes, comprising contacting a CTLprecursor lymphocyte with an antigen presenting cell that is expressinga polynucleotide coding for a polypeptide of the invention and whereinsaid polynucleotide is operably linked to a promoter.

A variety of techniques exist for assaying the activity of CTL. Suchassays are well-known in the art and their selection is left to theskilled artisan. CTLs are known to release, induce, increase, enhance,stimulate or activate expression or production of a cytokine. Assayselection is left to the skilled artisan.

After expansion of the antigen-specific CTLs, the CTLs are thenadoptively transferred back into the patient, where they will destroytheir specific target cell. Methodologies for reinfusing T cells into apatient are well known and exemplified in U.S. Pat. No. 4,844,893 toHonski, et al., and U.S. Pat. No. 4,690,915 to Rosenberg.

The peptide-specific CTL can be purified from the stimulator cells priorto infusion into the patient. For example, monoclonal antibodiesdirected toward the cell surface protein CD8, present on CTL, can beused in conjunction with a variety of isolation techniques such asantibody panning, flow cytometric sorting, and magnetic bead separationto purify the peptide-specific CTL away from any remaining non-peptidespecific lymphocytes or from the stimulator cells.

Thus, one embodiment of the present invention relates to a process fortreating a subject with DV infection or DV exposure characterized byinfected cells expressing complexes of a molecule from A2, or A24, or B7supertypes, whereby CTLs produced in vitro according to the presentinvention are administered in an amount sufficient to destroy theinfected cells through direct lysis or to effect the destruction of theinfected cells indirectly through the elaboration of cytokines.

Another embodiment of the present invention is directed to a process fortreating a subject with infection characterized by infected cellsexpressing any class I MHC molecule and an epitope of SEQ ID NO: 1-17,whereby the CTLs are produced in vitro and are specific for the epitopeor original protein and are administered in an amount sufficient todestroy the infected cells through direct lysis or to effect thedestruction of the infected cells indirectly through the elaboration ofcytokines.

The ex vivo generated CTL can be used to identify and isolate the T cellreceptor molecules specific for the peptide. The genes encoding thealpha and beta chains of the T cell receptor can be cloned into anexpression vector system and transferred and expressed in naive T cellsfrom peripheral blood, T cells from lymph nodes, or T lymphocyteprogenitor cells from bone marrow. These T cells, which would then beexpressing a peptide-specific T cell receptor, would then have anti-DVreactivity and could be used in adoptive therapy of DV infection, andmore specifically DV serotypes 1-4.

In addition to their use for therapeutic or prophylactic purposes, theimmunogenic peptides of the present invention are useful as screeningand diagnostic agents. Thus, the immunogenic peptides of the presentinvention, together with modern techniques of CTL screening, make itpossible to screen patients for the presence of T cells specific forthese peptides as a test for DV infection, exposure and immune response.The results of such screening may help determine the efficacy ofproceeding with the regimen of treatment disclosed herein using theimmunogens of the present invention.

The oligopeptides of the invention, such as SEQ ID NO: 1-17, can also beused to prepare class I MHC tetramers or pentamers which can be used inconjunction with flow cytometry to quantitate the frequency ofpeptide-specific CTL that are present in a sample of lymphocytes from anindividual. Specifically, for example, class I MHC molecules comprisingpeptides of SEQ ID NO: 1-17, would be combined to form tetramers asexemplified in U.S. Pat. No. 5,635,363. Said tetramers would find use inmonitoring the frequency of CTLs in the peripheral blood or lymph nodesof an individual who is vaccinated or undergoing immunotherapy with thepeptides, proteins, or polynucleotides of the invention, and it would beexpected that successful immunization would lead to an increase in thefrequency of the peptide-specific CTL.

Alternatively, the immunogenic peptides disclosed herein, as well asfunctionally similar homologs thereof, may be used to screen a samplefor the presence of CTLs that specifically recognize the correspondingepitopes. The lymphocytes to be screened in this assay will normally beobtained from the peripheral blood, but lymphocytes can be obtained fromother sources, including lymph nodes, spleen, and body fluids. Thepeptides of the present invention may then be used as a diagnostic toolto evaluate the efficacy of the immunotherapeutic treatments disclosedherein. Thus, the in vitro generation of CTL as described above would beused to determine if patients are likely to respond to the peptide invivo. Similarly, the in vitro generation of CTL cam be done with samplesof lymphocytes obtained from the patient before and after treatment withthe peptides. Successful generation of CTL in vivo should then berecognized by a correspondingly easier ability to generatepeptide-specific CTL in vitro from lymphocytes obtained followingtreatment in comparison to those obtained before treatment.

As stated above, a prophylactic or therapeutic vaccine in accordancewith the present invention may include one or more of the hereinabovedescribed polypeptides or active fragments thereof, or a composition, orpool, of immunogenic peptides disclosed herein. When employing more thanone polypeptide or active fragment, such as two or more polypeptidesand/or active fragments may be used as a physical mixture or as a fusionof two or more polypeptides or active fragments. The fusion fragment orfusion polypeptide may be produced, for example, by recombinanttechniques or by the use of appropriate linkers for fusing previouslyprepared polypeptides or active fragments.

The immunogenic molecules of the invention, including vaccinecompositions, may be utilized according to the present invention forpurposes of preventing, suppressing or treating diseases causing theexpression of the immunogenic peptides disclosed herein, such as wherethe antigen is being expressed by DV infected cells. As used inaccordance with the present invention, the term “prevention” relates toa process of prophylaxis in which an animal, especially a mammal, andmost especially a human, is exposed to an immunogen of the presentinvention prior to the induction or onset of the disease process. Thiscould be done where an individual is at high risk for DV infection basedon the living or travel to the DV endemic areas. Alternatively, theimmunogen could be administered to the general population as isfrequently done for any infectious diseases. Alternatively, the term“suppression” is often used to describe a condition wherein the diseaseprocess has already begun but obvious symptoms of said condition haveyet to be realized. Thus, the cells of an individual may have beeninfected but no outside signs of the disease have yet been clinicallyrecognized. In either case, the term prophylaxis can be applied toencompass both prevention and suppression. Conversely, the term“treatment” is often utilized to mean the clinical application of agentsto combat an already existing condition whose clinical presentation hasalready been realized in a patient. This would occur where an individualhas already been diagnosed as having confirmed DV infection.

It is understood that the suitable dosage of an immunogen of the presentinvention will depend upon the age, sex, health, and weight of therecipient, the kind of concurrent treatment, if any, the frequency oftreatment, and the nature of the effect desired. However, the mostpreferred dosage can be tailored to the individual subject, asdetermined by the researcher or clinician. The total dose required forany given treatment will commonly be determined with respect to astandard reference dose as set by a manufacturer, such as is commonlydone with vaccines, such dose being administered either in a singletreatment or in a series of doses, the success of which will depend onthe production of a desired immunological result (i.e., successfulproduction of a CTL-mediated response to the antigen, which responsegives rise to the prevention and/or treatment desired).

The therapeutically effective amount of a composition containing one ormore of the immunogens of this invention, is an amount sufficient toinduce an effective CTL response to prevent, cure or arrest diseaseprogression. Thus, this dose will depend, among other things, on theidentity of the immunogens used, the nature of the disease condition,the severity of the disease condition, the extent of any need to preventsuch a condition where it has not already been detected, the manner ofadministration dictated by the situation requiring such administration,the weight and state of health of the individual receiving suchadministration, and the sound judgment of the clinician or researcher.Thus, for purposes of prophylactic or therapeutic administration,effective amounts would generally lie within the range of from 1.0 μg toabout 5,000 μg of peptide for a 70 kg patient, followed by boostingdosages of from about 1.0 μg to about 1,000 μg of peptide pursuant to aboosting regimen over days, weeks or months, depending on therecipient's response and as necessitated by subsequent monitoring ofCTL-mediated activity within the bloodstream. Of course, such dosagesare to be considered only a general guide and, in a given situation, maygreatly exceed such suggested dosage regimens where the clinicianbelieves that the recipient's condition warrants more aggressiveadministration schedule. The efficacy of administering additional doses,and of increasing or decreasing the interval, may be re-evaluated on acontinuing basis, in view of the recipient's immunocompetence (forexample, the level of CTL activity with respect to acute or chronic DVinfection).

For such purposes, the immunogenic compositions according to the presentinvention may be used against a DV infection by administration to anindividual by a variety of routes. The composition may be administeredparenterally or orally, and, if parenterally, either systemically ortopically. Parenteral routes include subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, intranasal, transdermal, orbuccal routes. One or more such routes may be employed. Parenteraladministration can be, for example, by bolus injection or by gradualperfusion over time.

Generally, vaccines are prepared as injectables, in the form of aqueoussolutions or suspensions. Pharmaceutical carriers, diluents andexcipients can be generally added that are compatible with the activeingredients and acceptable for pharmaceutical use.

The concentration of the CTL stimulatory peptides of the invention inpharmaceutical formulations are subject to wide variation, includinganywhere from less than 0.01% by weight to as much as 50% or more.Factors such as volume and viscosity of the resulting composition mustalso be considered. The solvents, or diluents, used for suchcompositions include water, dimethylsulfoxide, PBS (phosphate bufferedsaline), or saline itself, or other possible carriers or excipients.

Aerosol administration is also an alternative, requiring only that theimmunogens be properly dispersed within the aerosol propellant. The useof a surfactant to properly disperse the immunogen may be required.Representative surfactants include caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. The surfactant mayconstitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The peptides and polypeptides of the invention may also be deliveredwith an adjuvant. Adjuvant effects can also be obtained by injecting avariety of cytokines along with the immunogens of the invention.

The peptides and polypeptides of the invention can also be added toprofessional antigen presenting cells such as dendritic cells that havebeen prepared ex vivo.

The present invention is also directed to a vaccine in which animmunogen of the present invention is delivered or administered in theform of a polynucleotide encoding a polypeptide or active fragment asdisclosed herein, whereby the peptide or polypeptide or active fragmentis produced in vivo. The polynucleotide may be included in a suitableexpression vector and combined with a pharmaceutically acceptablecarrier. A wide variety of vectors are available and apparent to thoseskilled in the art. Vaccinia vectors and methods useful in immunizationprotocols are described in U.S. Pat. No. 4,722,848, the disclosure ofwhich is incorporated herein by reference in its entirety.

Regardless of the nature of the composition given, additional vaccinecompositions may also accompany the immunogens of the present invention.Thus, for purposes of preventing or treating DV infection (e.g.,prophylactic or therapeutic vaccine), compositions containing theimmunogens disclosed herein may, in addition, contain other vaccinepharmaceuticals. The use of such compositions with multiple activeingredients is left to the discretion of the clinician.

While examples are provided to illustrate the invention, it is to beunderstood that these examples in no way limit the invention to theembodiments described herein and that other embodiments and uses will nodoubt suggest themselves to those skilled in the art. All publications,patents, and patent applications cited herein are hereby incorporated byreference, as are the references cited therein. It is also to beunderstood that throughout this disclosure where the singular is used,the plural may be inferred and vice versa and use of either is not to beconsidered limiting.

EXAMPLE

Dengue virus serotype 2 (DV2) (strain-16681), as well as DV type 3 (DV3)(strain-16562), were propagated in Vero cells and collected at 4 dayspost infection (dpi). Titer was determined using a plaque assay in Verocells. Thai isolates of all four DV serotypes were propagated. Allinfections were carried out at five MOI for 1 hr, after which, virus wasremoved and cells were incubated for additional 48-72 hrs.

HepG2, hepatoma cells, JY, EBV transformed lymphoblastoid B cells, T2,lymphoblasts, and K562, human leukemia cells were obtained from ATCC.HepG2 were maintained in DMEM:F12 medium while JY, T2, and K562 weremaintained in RPMI 1640 (Mediatech. Manassas, Va.) supplemented with 10%fetal bovine serum, and maintained at 37° C. in a humidified incubatorwith 5% CO2.

Dendritic cells (DC) were generated from leukopheresis obtained fromHLA-A2+ healthy donors (Research Blood Components, LLC, Brighton,Mass.).

Cell lysates from uninfected and Dengue-infected cell lines were testedfor dengue E protein expression by western blot analysis. Infected cellswere also analyzed for Dengue virus E protein using standardimmunohistochemical methods.

JY cells and DCs were infected with DV2 and DV3 respectively at fiveMOI. After a 1 hr pulse, virus was washed away, and cells were incubatedfor 72 hrs at 37° C. before harvested and processed for MHC peptideanalysis.

Cell lysates were prepared from dengue infected cells and MHC/peptidecomplexes were isolated by immunoaffinity chromatography using MHCmolecule specific antibodies The peptides purified from the MHCmolecules were fractionated using C-18 reversed phase (RP) column (4.6mm diameter×150 mm length) using an offline HPLC (Dionex, Sunnyvale,Calif.). The peptide containing fractions were collected and dried to 6μL under vacuum for LC/MS/MS analysis.

Mass spectrometry experiments were carried out using LTQ (Thermo) andOrbitrap instruments interfaced with nano ultimate HPLC (Dionex).RP-HPLC purified peptide fractions were injected individually into theLC-MS/MS system to identify the sequences of the peptides. The peptideswere analyzed using a Data-Dependent method. The acquired spectra datawere searched against Dengue (DV 1-4 serotypes) protein database usingProteome Discoverer (Thermo) to interpret data and derive peptidesequences.

Synthetic peptides were made and subjected to LC-MS/MS analysis underidentical experimental conditions as described above and their sequenceswere confirmed based on their MS/MS data. Candidate peptide sequenceswere confirmed by comparison of their MS/MS spectra with that of theirsynthetic analogs.

Heparinized blood from healthy HLA-A2+ donors was purchased fromResearch Blood Components, LLC (Brighton, Mass.). Peripheral bloodmononuclear cells (PBMC) were purified using differential centrifugationfollowing standard methods. PBMC were used to generate peptide specificCTL as described previously (James S. Testa, et al. J Infect Dis 205(4):647-655. (2012)).

Antigen stimulated interferon-γ (IFN-γ) release as a measure of CTLactivation was assayed using an ELISPOT assay kit (BD-Pharmingen, SanJose, Calif.) according to the manufacturer's instructions. Results arepresented as the number of interferon-γ producing cells per 1E6 PBMCs.Each assay was performed with PBMC from at least three different healthyHLA-A2+ donors. Error bars represent SEM of experimental replicates.

Seventeen epitopes including HLA-A2, A24, B7 and HLA-DR specific motifswere identified (Table 2) and four HLA-A2 specific epitopes (Seq ID: 1,2, 3 and 15) were selected for CTL characterization. Synthetic peptideswere made and used for CTL analysis. Table 2:

MHC Class I Associated T Cell Epitopes Presented by the Dengue VirusInfected Cells

Seq ID Peptide Protein Accession ID  1 NIQTAINQV NS4B Q9WDA6  2VTLLCLIPTV Capsid C Q2YHF2  3 TITEEIAVQ NS4B P29990  4 VLGWLEKYGV NS5gi158851822  5 ILGGLTWM NS2A gi148828521  6 KILIGSVITW Envelopegi259157612 glycoprotein  7 LFLGFTVQADI Envelope gi28171600 glycoprotein 8 LFGKGGIVTR Glycoprotein gi1000739  9 SPSRLASAI NS1 gi28171290 10IPSENEVKL NS1 gi224383594 11 NIQVAINQV NS4B P33478 12 NIQAAINQV NS4BP29990 13 VTLYLGVMV Capsid C P27912 14 VTLVLVGIV Capsid C P29991 15KLAEAIFKL NS5 P29990 16 LMWKQVTPELNHILS NS1 gi28171290 17 AGPLVAGGMLIACYNS2B gi239840450Productive Infection with Dengue Virus Strains

We determined the infectivity of JY, HepG2 cells and primary human DCsfrom HLA-A*0201+ donors, which possess high levels of the MHC class Imolecule that is most globally prevalent, HLA-A molecule. The cells wereinfected with DV2 and DV3, and expression of the envelope E protein wasdemonstrated (FIG. 1A). In addition, expression of E protein using animmunohistochemical method was demonstrated for HepG2 cells (FIG. 1B).

Identification of MHC Class I Presented Peptidec by Nano-LYCMS/MSAnalysis

MHC class I associated peptides isolated from dengue virus infectedcells were subjected to LC/MS/MS analysis to identify the peptides andtheir corresponding proteins. Employing this strategy, we identifiedseventeen MHC associated peptides. Seq ID: 1-15 represents MHC class Ipeptide of HLA-A2, A24 and B7 supertypes and Seq ID: 16 &17 representsMHC class 11 specific peptides. Prior to CTL characterizationexperiments, we confirmed the authenticity of four HLA-A2 specificpeptides (Seq ID: 1-[P4], 2-[P2], 3-[P3], 15-[P1]) using their syntheticpeptide analogs. The results of validation experiments on these 4peptides and their MS/MS spectra are shown in FIG. 2.

Cytotoxic T Cells Generated Against Dengue Virus Epitopes RecognizePeptide Loaded Targets as Well as DV-Infected Cells

In order to characterize the functionality of the identified DVepitopes, we generated cytotoxic T cells from HLA-A2+ healthy donorsusing synthetic peptides corresponding to the identified epitopes. Thecultures were tested in overnight ELISpot assays to measure IFNγ releasefrom activated CTLs. As shown in FIG. 3A, CTLs generated against all 4DV peptides recognize T2 cells loaded with the corresponding syntheticpeptide. Next, to confirm CTL activation against DV-infected targets, JYand HepG2 cells were infected with both DV2 and DV3 virus and used astargets in CTL ELISpot assays. As expected, all DV-infected cellsactivated CTL to secrete IFNγ (FIG. 3B). In addition, K562 cells, aknown NK cell target, were used as targets to measure non-specific IFNγsecretion.

Epitope-Specific CTLs Generated from DV-Infected Cells Recognize CellsInfected with Thai DV Isolates

We tested CTLs generated against the 4 epitopes using targets infectedwith all four DV serotypes. We infected HepG2 cells (FIG. 4A) or JYcells (FIG. 4B) with DV1-4 Thai isolates and verified proteinexpression. When infected HepG2 cells were used as targets in a CTLassay, all four epitope-specific CTLs were activated against all fourserotypes, although not to the same degree (FIG. 4C). In addition toHepG2 cells, we also tested professional APC JY cells, as targets. Asillustrated in FIG. 4D, most infected targets activate peptide-specificCTL. However, Seq ID 15 [P1] or Seq ID: 2 [P2] specific CTL recognizedonly DV2-infected targets.

Although the present invention has been described with reference tospecific embodiments, workers skilled in the art will recognize thatmany variations may be made therefrom, for example in the particularexperimental conditions herein described, and it is to be understood andappreciated that the disclosures in accordance with the invention showonly some preferred embodiments and objects and advantages of theinvention without departing from the broader scope and spirit of theinvention. It is to be understood and appreciated that these discoveriesin accordance with this invention are only those which are illustratedof the many additional potential applications that may be envisioned byone of ordinary skill in the art, and thus are not in any way intendedto be limiting of the invention. Accordingly, other objects andadvantages of the invention will be apparent to those skilled in the artfrom the detailed description together with the claims.

SEQUENCE LISTING

Accession Seq ID No Peptide Protein ID Seq ID No 1 NIQTAINQV NS4B Q9WDA6Seq ID No 2 VTLLCLIPTV Capsid C Q2YHF2 Seq ID No 3 TITEEIAVQ NS4B P29990Seq ID No 4 VLGWLEKYGV NS5 gi158851822 Seq ID No 5 ILGGLTWM NS2Agi148828521 Seq ID No 6 KILIGSVITW Envelope gi259157612 glyco- proteinSeq ID No 7 LFLGFTVQADI Envelope gi28171600 glyco- protein Seq ID No 8LFGKGGIVTR Glyco- gi1000739 protein Seq ID No 9 SPSRLASAI NS1 gi28171290Seq ID No 10 IPSENEVKL NS1 gi224383594 Seq ID No 11 NIQVAINQV NS4BP33478 Seq ID No 12 NIQAAINQV NS4B P29990 Seq ID No 13 VTLYLGVMVCapsid C P27912 Seq ID No 14 VTLVLVGIV Capsid C P29991 Seq ID No 15KLAEAIFKL NS5 P29990 Seq ID No 16 LMWKQVTPELNHILS NS1 gi28171290Seq ID No 17 AGPLVAGGMLIACY NS2B gi239840450

I claim:
 1. A method for inducing an immune response to dengue virusinfection in a subject comprising administering an immunogeniccomposition consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or 17 wherein said amino acid sequence binds to class IMHC molecules to induce a cytotoxic T lymphocyte-mediated immuneresponse.
 2. The method of claim 1 further comprising a pharmaceuticallyacceptable carrier.
 3. The method of claim 2 wherein thepharmaceutically acceptable carrier is an adjuvant.
 4. The method ofclaim 1 wherein said composition is a peptide, oligopeptide, orpolypeptide containing said amino acid sequence.
 5. The method of claim4 wherein said composition contains said amino acid sequence obtainedfrom a fragmented and processed peptide, oligopeptide, or polypeptide.6. The method of claim 1 wherein said amino acid sequence comprises atleast one T cell epitopic peptide.
 7. The method of claim 1 wherein saidamino acid sequence differs by no more than at least one amino acid fromSEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or
 17. 8.The method of claim 7 where the one amino acid difference is aconservative amino acid substitution.
 9. The method of claim 7 where theamino acid difference is a substitution of one hydrophobic amino acidwith another hydrophobic amino acid.
 10. The method of claim 1 whereinsaid cytotoxic T lymphocyte-mediated immune response is against at leastone serotype specific dengue virus infection.
 11. The method of claim 10wherein said cytotoxic T lymphocyte-mediated immune response is againstserotype specific dengue virus infections from a group consisting ofserotype 1, 2, 3, 4, and combinations thereof.
 12. The method of claim11 wherein said cytotoxic T lymphocyte-mediated immune response isagainst serotypes 1, 2, 3, and
 4. 13. A method for inducing a cytotoxicT lymphocyte-mediated immune response in a subject comprisingadministering an effective amount of a composition consisting of SEQ IDNO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or
 17. 14. Themethod of claim 13 wherein said composition is a peptide, oligopeptide,or polypeptide containing said amino acid sequence.
 15. The method ofclaim 14 wherein said composition contains said amino acid sequenceobtained from a fragmented and processed peptide, oligopeptide, orpolypeptide.
 16. The method of claim 13 wherein said amino acid sequencecomprises at least one epitopic peptide.
 17. The method of claim 13wherein said amino acid sequence differs by no more than at least oneamino acid from SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or
 17. 18. The method of claim 17 where the one amino aciddifference is a conservative amino acid substitution.
 19. The method ofclaim 17 where the amino acid difference is a substitution of onehydrophobic amino acid with another hydrophobic amino acid.
 20. A methodof preventing disease caused by dengue virus in a subject comprisingadministering an effective amount of a vaccine consisting of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 to thesubject.
 21. A method of inducing in a subject an immune response to atleast one dengue virus comprising administering to a subject in needthereof an amount of polynucleotide effective to induce an immuneresponse to at least one dengue virus serotype wherein saidpolynucleotide sequence encodes a polypeptide sequence that consists ofat least 95% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, or 17, wherein said amino acid sequence binds toclass I MHC molecules to induce a cytotoxic T lymphocyte-mediated immuneresponse.